The periodontal pathogen, Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, expresses several complex multi-gene cytotoxin systems that negatively impact specific types of cells important to the health of the human periodontium. These systems include genetic loci for a leukotoxin and a cytolethal distending toxin (Cdt). This impressive repertoire of virulence factors has not been found in other oral bacterial pathogens. The destruction caused by bacterial cytotoxins alters the local environment leading to increased microbial colonization and host inflammation. Our prior studies demonstrated that strains of A. actinomycetemcomitans isolated from subjects that have converted from a healthy to diseased periodontal state produce a Cdt that causes the growth arrest of primary human oral epithelial cells at the G2/M interphase of the cell cycle. This Cdt is an atypical AB type toxin composed of three gene products. The active subunit, CdtB, exhibits a DNA damaging activity that is reminiscent of mammalian type I deoxyribonucleases. CdtA, possibly in concert with CdtC, binds the toxin to the cell surface. The objectives of this application are to understand the structure/function relationships of the three Cdt subunits and to define the specific interactions of the subunits with primary differentiated and undifferentiated human gingival epithelial cells (HGEC). The general hypothesis of our study is that specific, unique functional domains in each of the three Cdt subunits contribute to the exquisite sensitivity and target specificity of epithelial cells. The primary role of CdtA is to recognize and bind to the cell surface receptor for the toxin while that of CdtC is to stabilize CdtB binding in the heterotrimer complex and to facilitate the intracellular transport of this subunit. In the context of colonization and pathogenesis, undifferentiated HGEC present a more likely target for the Cdt than differentiated HGEC. This application contains the following specific aims: (1) to identify and characterize specific domains in the CdtA and CdtC subunits required for heterotoxin assembly and binding to susceptible host cells, (2) to assess the contributions of the CdtC subunit in the assembly, intracellular transport and cytotoxicity of CdtB, (3) to use the cdt subunit mutants and Chinese hamster ovary (CHO) cell mutants to obtain detailed information about specific subunit functions and interactions in vivo and (4) to develop and begin to characterize a primary human gingival epithelial cell (HGEC) model to assess the effects of the Cdt. We expect that our approach will advance our ongoing structure/function studies of the Cdt and its possible role in bacterial virulence by providing new detailed information about holotoxin assembly, mechanism(s) of toxicity in HGEC and cellular interactions that can be exploited therapeutically to block Cdt activity to reduce the severity of the tissue destruction that is a hallmark of periodontal disease.